Gary J. Stephens

Differential expression of voltage-activated Na+ currents in two prostatic tumour cell lines: contribution to invasiveness in vitro

The voltage‐gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole‐cell patch clamp technique. The highly metastatic Mat‐Ly‐Lu cells expressed a transient, inward Na+ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT‐2 cells. Although both cell lines expressed a sustained, outward K+ current, this occurred at a significantly higher density in the AT‐2 than in the Mat‐Ly‐Lu cells. Incubation of the Mat‐Ly‐Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT‐2 cells.

Cannabidiol Displays Antiepileptiform and Antiseizure Properties In Vitro and In Vivo

Plant-derived cannabinoids (phytocannabinoids) are compounds with emerging therapeutic potential. Early studies suggested that cannabidiol (CBD) has anticonvulsant properties in animal models and reduced seizure frequency in limited human trials. Here, we examine the antiepileptiform and antiseizure potential of CBD using in vitro electrophysiology and an in vivo animal seizure model, respectively. CBD (0.01–100 μM) effects were assessed in vitro using the Mg2+-free and 4-aminopyridine (4-AP) models of epileptiform activity in hippocampal brain slices via multielectrode array recordings. In the Mg2+-free model, CBD decreased epileptiform local field potential (LFP) burst amplitude [in CA1 and dentate gyrus (DG) regions] and burst duration (in all regions) and increased burst frequency (in all regions). In the 4-AP model, CBD decreased LFP burst amplitude (in CA1 only at 100 μM CBD), burst duration (in CA3 and DG), and burst frequency (in all regions). CBD (1, 10, and 100 mg/kg) effects were also examined in vivo using the pentylenetetrazole model of generalized seizures. CBD (100 mg/kg) exerted clear anticonvulsant effects with significant decreases in incidence of severe seizures and mortality compared with vehicle-treated animals. Finally, CBD acted with only low affinity at cannabinoid CB1 receptors and displayed no agonist activity in [35S]guanosine 5′-O-(3-thio)triphosphate assays in cortical membranes. These findings suggest that CBD acts, potentially in a CB1 receptor-independent manner, to inhibit epileptiform activity in vitro and seizure severity in vivo. Thus, we demonstrate the potential of CBD as a novel antiepileptic drug in the unmet clinical need associated with generalized seizures.

Phytocannabinoids as novel therapeutic agents in CNS disorders

The Cannabis sativa herb contains over 100 phytocannabinoid (pCB) compounds and has been used for thousands of years for both recreational and medicinal purposes. In the past two decades, characterisation of the bodys endogenous cannabinoid (CB) (endocannabinoid, eCB) system (ECS) has highlighted activation of central CB(1) receptors by the major pCB, Δ(9)-tetrahydrocannabinol (Δ(9)-THC) as the primary mediator of the psychoactive, hyperphagic and some of the potentially therapeutic properties of ingested cannabis. Whilst Δ(9)-THC is the most prevalent and widely studied pCB, it is also the predominant psychotropic component of cannabis, a property that likely limits its widespread therapeutic use as an isolated agent. In this regard, research focus has recently widened to include other pCBs including cannabidiol (CBD), cannabigerol (CBG), Δ(9)tetrahydrocannabivarin (Δ(9)-THCV) and cannabidivarin (CBDV), some of which show potential as therapeutic agents in preclinical models of CNS disease. Moreover, it is becoming evident that these non-Δ(9)-THC pCBs act at a wide range of pharmacological targets, not solely limited to CB receptors. Disorders that could be targeted include epilepsy, neurodegenerative diseases, affective disorders and the central modulation of feeding behaviour. Here, we review pCB effects in preclinical models of CNS disease and, where available, clinical trial data that support therapeutic effects. Such developments may soon yield the first non-Δ(9)-THC pCB-based medicines.

Hyperpolarization-activated currents in presynaptic terminals of mouse cerebellar basket cells

1 Using patch‐clamp techniques, a hyperpolarization‐activated current (Ih) was recorded from synaptic terminals of mouse cerebellar basket cells. 2 I h was blocked quickly and reversibly by 2 mM Cs+, and subtraction revealed a rapidly activating and deactivating Ih current. Similar gating and block of presynaptic Ih were also seen with the more selective inhibitor ZD 7288 (10 μM). 3 The time constant of activation (τa) of presynaptic Ih current became faster with membrane hyperpolarization, being ≈74 ms at ‐130 mV, changing e‐fold for a 33 mV change in membrane potential. 4 Whole‐cell recordings from basket cell somata also revealed an Ih current, which was similarly sensitive to block by ZD 7288. 5 Inhibition of Ih by 10 μM ZD 7288 reduced the frequency (≈34 %) and amplitude (≈26 %) of spontaneous IPSCs (sIPSCs) recorded in Purkinje cells, one of the principal synaptic targets of basket neurones. 6 This is the first report of an Ih current in mammalian inhibitory presynaptic terminals, which may be an important target for neuromodulation in the cerebellum. Comparing the biophysical properties and distribution of cloned hyperpolarization‐activated cation channels, we also suggest a molecular candidate underlying Ih at these synapses.

Cannabidiol exerts anti-convulsant effects in animal models of temporal lobe and partial seizures

Cannabis sativa has been associated with contradictory effects upon seizure states despite its medicinal use by numerous people with epilepsy. We have recently shown that the phytocannabinoid cannabidiol (CBD) reduces seizure severity and lethality in the well-established in vivo model of pentylenetetrazole-induced generalised seizures, suggesting that earlier, small-scale clinical trials examining CBD effects in people with epilepsy warrant renewed attention. Here, we report the effects of pure CBD (1, 10 and 100mg/kg) in two other established rodent seizure models, the acute pilocarpine model of temporal lobe seizure and the penicillin model of partial seizure. Seizure activity was video recorded and scored offline using model-specific seizure severity scales. In the pilocarpine model CBD (all doses) significantly reduced the percentage of animals experiencing the most severe seizures. In the penicillin model, CBD (≥ 10 mg/kg) significantly decreased the percentage mortality as a result of seizures; CBD (all doses) also decreased the percentage of animals experiencing the most severe tonic-clonic seizures. These results extend the anti-convulsant profile of CBD; when combined with a reported absence of psychoactive effects, this evidence strongly supports CBD as a therapeutic candidate for a diverse range of human epilepsies.

Nonpsychotropic Plant Cannabinoids, Cannabidivarin (CBDV) and Cannabidiol (CBD), Activate and Desensitize Transient Receptor Potential Vanilloid 1 (TRPV1) Channels in Vitro: Potential for the Treatment of Neuronal Hyperexcitability

Epilepsy is the most common neurological disorder, with over 50 million people worldwide affected. Recent evidence suggests that the transient receptor potential cation channel subfamily V member 1 (TRPV1) may contribute to the onset and progression of some forms of epilepsy. Since the two nonpsychotropic cannabinoids cannabidivarin (CBDV) and cannabidiol (CBD) exert anticonvulsant activity in vivo and produce TRPV1-mediated intracellular calcium elevation in vitro, we evaluated the effects of these two compounds on TRPV1 channel activation and desensitization and in an in vitro model of epileptiform activity. Patch clamp analysis in transfected HEK293 cells demonstrated that CBD and CBDV dose-dependently activate and rapidly desensitize TRPV1, as well as TRP channels of subfamily V type 2 (TRPV2) and subfamily A type 1 (TRPA1). TRPV1 and TRPV2 transcripts were shown to be expressed in rat hippocampal tissue. When tested on epileptiform neuronal spike activity in hippocampal brain slices exposed to a Mg(2+)-free solution using multielectrode arrays (MEAs), CBDV reduced both epileptiform burst amplitude and duration. The prototypical TRPV1 agonist, capsaicin, produced similar, although not identical effects. Capsaicin, but not CBDV, effects on burst amplitude were reversed by IRTX, a selective TRPV1 antagonist. These data suggest that CBDV antiepileptiform effects in the Mg(2+)-free model are not uniquely mediated via activation of TRPV1. However, TRPV1 was strongly phosphorylated (and hence likely sensitized) in Mg(2+)-free solution-treated hippocampal tissue, and both capsaicin and CBDV caused TRPV1 dephosphorylation, consistent with TRPV1 desensitization. We propose that CBDV effects on TRP channels should be studied further in different in vitro and in vivo models of epilepsy.

Calcium Channel β Subunit Promotes Voltage-Dependent Modulation of α1B by Gβγ

Abstract Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming α 1 subunit and auxiliary subunits, including the intracellular β subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G βγ dimer concentration. Here we have examined the interaction between the regulation of N-type ( α 1B) channels by their β subunits and by G βγ dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC β subunit, we found a significantly larger G βγ -dependent inhibition of α 1B channel activation when the VDCC α 1B and β subunits were coexpressed. In the absence of coexpressed VDCC β subunit, the G βγ dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC β subunit coexpression restored all of the hallmarks of G βγ modulation. These results suggest that the VDCC β subunit must be present for G βγ to induce voltage-dependent modulation of N-type calcium channels.

CB1 Receptor Allosteric Modulators Display Both Agonist and Signaling Pathway Specificity

We have previously identified allosteric modulators of the cannabinoid CB1 receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB1 receptor agonist [3H]CP55940 but producing a decrease in CB1 receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB1 receptor activation. We assessed the effect of these compounds on CB1 receptor agonist–induced [35S]GTPγS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and β-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB1 agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signaling as compared with WIN55212 and having little effect on [3H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding, simulation (Gαs-mediated), and inhibition (Gαi-mediated) of cAMP production and β-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic-binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB1 agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.

The α1B Ca2+ channel amino terminus contributes determinants for β subunit‐mediated voltage‐dependent inactivation properties

1 Co‐expression of auxiliary β subunits with the α1B Ca2+ channel subunit in COS‐7 cells resulted in an increase in current density and a hyperpolarising shift in the mid‐point of activation. Amongst the β subunits, β2a in particular, but also β4 and β1b caused a significant retardation of the voltage‐dependent inactivation compared to currents with α1B alone, whilst no significant changes in inactivation properties were seen for the β3 subunit in this system. 2 Prevention of β2a palmitoylation, by introducing cysteine to serine mutations (β2a(C3,4S)), greatly reduced the ability of β2a to retard voltage‐dependent inactivation. 3 Deletion of the proximal half of the α1B cytoplasmic amino terminus (α1BΔ1‐55) differentially affected β subunit‐mediated voltage‐dependent inactivation properties. These effects were prominent with the β2a subunit and, to a lesser extent, with β1b. For β2a, the major effects of this deletion were a partial reversal of β2a‐mediated retardation of inactivation and the introduction of a fast component of inactivation, not seen with full‐length α1B. Deletion of the amino terminus had no other major effects on the measured biophysical properties of α1B when co‐expressed with β subunits. 4 Transfer of the whole α1B amino terminus into α1C (α1bCCCC) conferred a similar retardation of inactivation on α1C when co‐expressed with β2a to that seen in parental α1B. 5 Individual (α1B(Q47A) and α1B(R52A)) and double (α1B(R52,54A)) point mutations within the amino terminus of α1B also opposed the β2a‐mediated retardation of α1B inactivation kinetics. 6 These results indicate that the α1B amino terminus contains determinants for β subunit‐mediated voltage‐dependent inactivation properties. Furthermore, effects were β subunit selective. As deletion of the α1B amino terminus only partially opposed β subunit‐mediated changes in inactivation properties, the amino terminus is likely to contribute to a complex site necessary for complete β subunit function.

Cannabidivarin is anticonvulsant in mouse and rat

Phytocannabinoids in Cannabis sativa have diverse pharmacological targets extending beyond cannabinoid receptors and several exert notable anticonvulsant effects. For the first time, we investigated the anticonvulsant profile of the phytocannabinoid cannabidivarin (CBDV) in vitro and in in vivo seizure models.